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1.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204449

RESUMO

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including ß-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR-/- mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR-/- mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.


Assuntos
Proteínas de Arcabouço Homer/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Osteoblastos/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Cálcio/metabolismo , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas , Feminino , Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Detecção de Cálcio/genética
2.
Rinsho Ketsueki ; 62(5): 512-520, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-34248129

RESUMO

In human hematopoiesis, cells of various lineages exist, such as neutrophils, lymphocytes, and erythrocytes. Unveiling the pathway from stem cells to the various lineages helps us understand the blood disorders and develop therapies for them. We have studied the developmental pathway of hematopoiesis for decades and found that myeloid potential is retained just before the differentiation into each lineage of the various lineage progenitors. This uniqueness of myeloid cells might reflect the character of mixed-phenotype leukemia and provide a very important clue in determining the evolutional history of blood cells. Recent studies concerning the differentiation pathways of megakaryocytes and granulocytes as well as the findings on the hemocytes of invertebrates have strongly supported the concept of the uniqueness of myeloid cells and enabled us to propose insights into the evolutional history of blood. In this paper, we discuss the origin of blood cells in the context of developmental pathways during ontogeny and phylogeny.


Assuntos
Hematopoese , Células Mieloides , Diferenciação Celular , Linhagem da Célula , Granulócitos , Humanos
3.
Nat Commun ; 12(1): 3450, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103504

RESUMO

The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Chaperonas de Histonas/metabolismo , Músculo Esquelético/patologia , Fatores de Transcrição/metabolismo , Acetilação , Animais , Proteínas de Ciclo Celular/deficiência , Linhagem Celular , Linhagem da Célula/genética , Loci Gênicos , Chaperonas de Histonas/deficiência , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Músculo Esquelético/fisiopatologia , Regeneração , Sequências Reguladoras de Ácido Nucleico/genética , Células Satélites de Músculo Esquelético/metabolismo , Fatores de Transcrição/deficiência
4.
Nat Commun ; 12(1): 3679, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140473

RESUMO

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.


Assuntos
Implantação do Embrião/genética , Desenvolvimento Embrionário , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Análise de Célula Única/métodos , Via de Sinalização Wnt , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Linhagem da Célula , Células Cultivadas , Implantação do Embrião/fisiologia , Embrião de Mamíferos , Fatores de Crescimento de Fibroblastos/metabolismo , Gastrulação/fisiologia , Camadas Germinativas/citologia , Humanos , Processamento de Imagem Assistida por Computador , Família Multigênica , Proteína Nodal/antagonistas & inibidores , RNA-Seq , Análise Espaço-Temporal
5.
Nat Commun ; 12(1): 3715, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140474

RESUMO

A comprehensive transcriptomic survey of pigs can provide a mechanistic understanding of tissue specialization processes underlying economically valuable traits and accelerate their use as a biomedical model. Here we characterize four transcript types (lncRNAs, TUCPs, miRNAs, and circRNAs) and protein-coding genes in 31 adult pig tissues and two cell lines. We uncover the transcriptomic variability among 47 skeletal muscles, and six adipose depots linked to their different origins, metabolism, cell composition, physical activity, and mitochondrial pathways. We perform comparative analysis of the transcriptomes of seven tissues from pigs and nine other vertebrates to reveal that evolutionary divergence in transcription potentially contributes to lineage-specific biology. Long-range promoter-enhancer interaction analysis in subcutaneous adipose tissues across species suggests evolutionarily stable transcription patterns likely attributable to redundant enhancers buffering gene expression patterns against perturbations, thereby conferring robustness during speciation. This study can facilitate adoption of the pig as a biomedical model for human biology and disease and uncovers the molecular bases of valuable traits.


Assuntos
Tecido Adiposo/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , RNA Circular/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Transcriptoma/genética , Processamento Alternativo , Animais , Evolução Biológica , Linhagem Celular , Linhagem da Célula , Núcleo Celular/genética , Núcleo Celular/metabolismo , Elementos Facilitadores Genéticos , Evolução Molecular , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , Mitocôndrias/metabolismo , Conformação Molecular , Miofibrilas/genética , Miofibrilas/metabolismo , Filogenia , Regiões Promotoras Genéticas , RNA Circular/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Análise Espacial , Suínos
6.
Nat Commun ; 12(1): 3708, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140506

RESUMO

3D genome alternations can dysregulate gene expression by rewiring enhancer-promoter interactions and lead to diseases. We report integrated analyses of 3D genome alterations and differential gene expressions in 18 newly diagnosed T-lineage acute lymphoblastic leukemia (T-ALL) patients and 4 healthy controls. 3D genome organizations at the levels of compartment, topologically associated domains and loop could hierarchically classify different subtypes of T-ALL according to T cell differentiation trajectory, similar to gene expressions-based classification. Thirty-four previously unrecognized translocations and 44 translocation-mediated neo-loops are mapped by Hi-C analysis. We find that neo-loops formed in the non-coding region of the genome could potentially regulate ectopic expressions of TLX3, TAL2 and HOXA transcription factors via enhancer hijacking. Importantly, both translocation-mediated neo-loops and NUP98-related fusions are associated with HOXA13 ectopic expressions. Patients with HOXA11-A13 expressions, but not other genes in the HOXA cluster, have immature immunophenotype and poor outcomes. Here, we highlight the potentially important roles of 3D genome alterations in the etiology and prognosis of T-ALL.


Assuntos
Cromossomos/metabolismo , Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Conformação Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfócitos T/metabolismo , Translocação Genética , Acetilação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Criança , Sequenciamento de Cromatina por Imunoprecipitação , Cromossomos/genética , Progressão da Doença , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/imunologia , Ontologia Genética , Hematopoese/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imunofenotipagem , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/mortalidade , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Prognóstico , Linfócitos T/patologia , Adulto Jovem
7.
Nat Commun ; 12(1): 3933, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168132

RESUMO

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.


Assuntos
Epigênese Genética , Células Epiteliais/citologia , Complexo Repressor Polycomb 2/genética , Timo/citologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Epiteliais/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Timócitos/citologia , Timócitos/fisiologia , Timo/fisiologia
8.
Nat Commun ; 12(1): 3851, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158501

RESUMO

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.


Assuntos
Padronização Corporal/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético/metabolismo , Somitos/metabolismo , Animais , Linhagem da Célula/genética , Células Cultivadas , Embrião de Galinha , Extremidades/embriologia , Fibroblastos/citologia , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somitos/citologia , Somitos/embriologia
9.
Nat Commun ; 12(1): 3876, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162856

RESUMO

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Homeostase/genética , Células-Tronco Mesenquimais/metabolismo , Regeneração/genética , Testículo/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Célula Única/métodos , Testículo/citologia
10.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072880

RESUMO

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Blastocisto/efeitos dos fármacos , Fator de Transcrição CDX2/genética , Lisofosfolipídeos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Aciltransferases/genética , Animais , Massa Celular Interna do Blastocisto/efeitos dos fármacos , Bovinos , Linhagem da Célula/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Trofoblastos/efeitos dos fármacos
11.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072061

RESUMO

Numerous studies demonstrate the essential role of mesenchymal stem cells (MSCs) in the treatment of metabolic and inflammatory diseases, as these cells are known to modulate humoral and cellular immune responses. In this manuscript, we efficiently present two novel approaches to obtain MSCs from equine or human sources. In our first approach, we used electro-acupuncture as previously described by our group to mobilize MSCs into the peripheral blood of horses. For equine MSC collection, culture, and expansion, we used the Miltenyi Biotec CliniMACS Prodigy system of automated cell manufacturing. Using this system, we were able to generate appoximately 100 MSC colonies that exhibit surface marker expression of CD105 (92%), CD90 (85%), and CD73 (88%) within seven days of blood collection. Our second approach utilized the iPSC embryoid bodies from healthy or diabetic subjects where the iPSCs were cultured in standard media (endothelial + mesoderm basal media). After 21 days, the cells were FACS sorted and exhibited surface marker expression of CD105, CD90, and CD73. Both the equine cells and the human iPSC-derived MSCs were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Both methods described simple and highly efficient methods to produce cells with surface markers phenotypically considered as MSCs and may, in the future, facilitate rapid production of MSCs with therapeutic potential.


Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Animais , Antígenos CD/metabolismo , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Imunofluorescência , Cavalos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo
12.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069546

RESUMO

Long non-coding RNAs (lncRNAs) regulate a diverse array of cellular processes at the transcriptional, post-transcriptional, translational, and post-translational levels. Accumulating evidence suggests that lncRNA MEG3 exerts a large repertoire of regulatory functions in cellular stemness. This review focuses on the molecular mechanisms by which lncRNA MEG3 functions as a signal, scaffold, guide, and decoy for multi-lineage differentiation and even cancer progression. The role of MEG3 in various types of stem cells and cancer stem cells is discussed. Here, we provide an overview of the functional versatility of lncRNA MEG3 in modulating pluripotency, differentiation, and cancer stemness.


Assuntos
RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Humanos , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/metabolismo
13.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1232098

RESUMO

Comprehensive and accurate comparisons of transcriptomic distributions of cells from samples taken from two different biological states, such as healthy versus diseased individuals, are an emerging challenge in single-cell RNA sequencing (scRNA-seq) analysis. Current methods for detecting differentially abundant (DA) subpopulations between samples rely heavily on initial clustering of all cells in both samples. Often, this clustering step is inadequate since the DA subpopulations may not align with a clear cluster structure, and important differences between the two biological states can be missed. Here, we introduce DA-seq, a targeted approach for identifying DA subpopulations not restricted to clusters. DA-seq is a multiscale method that quantifies a local DA measure for each cell, which is computed from its k nearest neighboring cells across a range of k values. Based on this measure, DA-seq delineates contiguous significant DA subpopulations in the transcriptomic space. We apply DA-seq to several scRNA-seq datasets and highlight its improved ability to detect differences between distinct phenotypes in severe versus mildly ill COVID-19 patients, melanomas subjected to immune checkpoint therapy comparing responders to nonresponders, embryonic development at two time points, and young versus aging brain tissue. DA-seq enabled us to detect differences between these phenotypes. Importantly, we find that DA-seq not only recovers the DA cell types as discovered in the original studies but also reveals additional DA subpopulations that were not described before. Analysis of these subpopulations yields biological insights that would otherwise be undetected using conventional computational approaches.


Assuntos
Envelhecimento/genética , COVID-19/genética , Linhagem da Célula/genética , Melanoma/genética , RNA Citoplasmático Pequeno/genética , Neoplasias Cutâneas/genética , Envelhecimento/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Encéfalo/citologia , Encéfalo/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Linhagem da Célula/imunologia , Citocinas/genética , Citocinas/imunologia , Conjuntos de Dados como Assunto , Células Dendríticas/imunologia , Células Dendríticas/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Melanoma/imunologia , Melanoma/patologia , Monócitos/imunologia , Monócitos/virologia , Fenótipo , RNA Citoplasmático Pequeno/imunologia , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Análise de Célula Única/métodos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/imunologia , Linfócitos T/virologia , Transcriptoma
14.
Mol Cell ; 81(12): 2583-2595.e6, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33961797

RESUMO

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.


Assuntos
Linhagem da Célula/genética , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Humanos , Ligação Proteica , Proteínas de Ligação a RNA/fisiologia , Domínio Tudor , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia
15.
J Vis Exp ; (170)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33999034

RESUMO

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.


Assuntos
Hematopoese , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Hibridização In Situ , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
16.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34001664

RESUMO

Comprehensive and accurate comparisons of transcriptomic distributions of cells from samples taken from two different biological states, such as healthy versus diseased individuals, are an emerging challenge in single-cell RNA sequencing (scRNA-seq) analysis. Current methods for detecting differentially abundant (DA) subpopulations between samples rely heavily on initial clustering of all cells in both samples. Often, this clustering step is inadequate since the DA subpopulations may not align with a clear cluster structure, and important differences between the two biological states can be missed. Here, we introduce DA-seq, a targeted approach for identifying DA subpopulations not restricted to clusters. DA-seq is a multiscale method that quantifies a local DA measure for each cell, which is computed from its k nearest neighboring cells across a range of k values. Based on this measure, DA-seq delineates contiguous significant DA subpopulations in the transcriptomic space. We apply DA-seq to several scRNA-seq datasets and highlight its improved ability to detect differences between distinct phenotypes in severe versus mildly ill COVID-19 patients, melanomas subjected to immune checkpoint therapy comparing responders to nonresponders, embryonic development at two time points, and young versus aging brain tissue. DA-seq enabled us to detect differences between these phenotypes. Importantly, we find that DA-seq not only recovers the DA cell types as discovered in the original studies but also reveals additional DA subpopulations that were not described before. Analysis of these subpopulations yields biological insights that would otherwise be undetected using conventional computational approaches.


Assuntos
Envelhecimento/genética , COVID-19/genética , Linhagem da Célula/genética , Melanoma/genética , RNA Citoplasmático Pequeno/genética , Neoplasias Cutâneas/genética , Envelhecimento/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Encéfalo/citologia , Encéfalo/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Linhagem da Célula/imunologia , Citocinas/genética , Citocinas/imunologia , Conjuntos de Dados como Assunto , Células Dendríticas/imunologia , Células Dendríticas/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Melanoma/imunologia , Melanoma/patologia , Monócitos/imunologia , Monócitos/virologia , Fenótipo , RNA Citoplasmático Pequeno/imunologia , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Análise de Célula Única/métodos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/imunologia , Linfócitos T/virologia , Transcriptoma
17.
Nat Commun ; 12(1): 2761, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980830

RESUMO

At numerous locations of the body, transition zones are localized at the crossroad between two types of epithelium and are frequently associated with neoplasia involving both type of tissues. These transition zones contain cells expressing markers of adult stem cells that can be the target of early transformation. The mere fact that transition zone cells can merge different architecture with separate functions implies for a unique plasticity that these cells must display in steady state. However, their roles during tissue regeneration in normal and injured state remain unknown. Here, by using in vivo lineage tracing, single-cell transcriptomics, computational modeling and a three-dimensional organoid culture system of transition zone cells, we identify a population of Krt17+ basal cells with multipotent properties at the squamo-columnar anorectal junction that maintain a squamous epithelium during normal homeostasis and can participate in the repair of a glandular epithelium following tissue injury.


Assuntos
Canal Anal/citologia , Homeostase , Reto/citologia , Regeneração , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Plasticidade Celular , Humanos , Mucosa Intestinal/citologia , Queratina-17/genética , Queratina-17/metabolismo , Camundongos , Organoides/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cicatrização
18.
Nat Commun ; 12(1): 2564, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963183

RESUMO

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-ß) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.


Assuntos
Células Endoteliais/metabolismo , Endotélio/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula , Endotélio/citologia , Feminino , Técnicas de Inativação de Genes , Proteínas Hedgehog/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno , Receptores Notch/metabolismo , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/genética
19.
Nat Commun ; 12(1): 2559, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963182

RESUMO

Multiple myeloma (MM) is characterized by the uncontrolled proliferation of plasma cells. Despite recent treatment advances, it is still incurable as disease progression is not fully understood. To investigate MM and its immune environment, we apply single cell RNA and linked-read whole genome sequencing to profile 29 longitudinal samples at different disease stages from 14 patients. Here, we collect 17,267 plasma cells and 57,719 immune cells, discovering patient-specific plasma cell profiles and immune cell expression changes. Patients with the same genetic alterations tend to have both plasma cells and immune cells clustered together. By integrating bulk genomics and single cell mapping, we track plasma cell subpopulations across disease stages and find three patterns: stability (from precancer to diagnosis), and gain or loss (from diagnosis to relapse). In multiple patients, we detect "B cell-featured" plasma cell subpopulations that cluster closely with B cells, implicating their cell of origin. We validate AP-1 complex differential expression (JUN and FOS) in plasma cell subpopulations using CyTOF-based protein assays, and integrated analysis of single-cell RNA and CyTOF data reveals AP-1 downstream targets (IL6 and IL1B) potentially leading to inflammation regulation. Our work represents a longitudinal investigation for tumor and microenvironment during MM progression and paves the way for expanding treatment options.


Assuntos
Linfócitos B/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Recidiva Local de Neoplasia/genética , Microambiente Tumoral/imunologia , Idoso , Linfócitos B/citologia , Linfócitos B/imunologia , Linhagem da Célula , Evolução Clonal/genética , Estudos de Coortes , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Haplótipos , Humanos , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Família Multigênica , Mieloma Múltiplo/sangue , Mieloma Múltiplo/patologia , Mutação , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/imunologia , Proteínas Proto-Oncogênicas c-fos/sangue , Proteínas Proto-Oncogênicas c-jun/sangue , RNA-Seq , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Análise de Célula Única
20.
Cell Stem Cell ; 28(6): 1016-1022.e4, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33957081

RESUMO

Human naive pluripotent cells can differentiate into extraembryonic trophectoderm and hypoblast. Here we describe a human embryo model (blastoid) generated by self-organization. Brief induction of trophectoderm leads to formation of blastocyst-like structures within 3 days. Blastoids are composed of three tissue layers displaying exclusive lineage markers, mimicking the natural blastocyst. Single-cell transcriptome analyses confirm segregation of trophectoderm, hypoblast, and epiblast with high fidelity to the human embryo. This versatile and scalable system provides a robust experimental model for human embryo research.


Assuntos
Blastocisto , Embrião de Mamíferos , Diferenciação Celular , Linhagem da Célula , Camadas Germinativas , Humanos , Células-Tronco
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